Filariid anti-P22U antibodies

ABSTRACT

The present invention relates to isolated parasitic helminth nucleic acid sequences capable of hybridizing, under stringent conditions, to at least a portion of  D. immitis  nucleic acid sequence p4 and/or to at least a portion of  D. immitis  nucleic acid sequence p22U; to isolated parasitic helminth proteins that are encoded by such parasitic helminth nucleic acid sequences and that are capable of selectively binding to at least one component of immune serum capable of inhibiting helminth development; and to antibodies raised against such isolated parasitic helminth proteins. The present invention also relates to therapeutic compositions comprising such isolated nucleic acid sequences, proteins and/or antibodies. The present invention also includes methods to produce and use such nucleic acids, proteins, antibodies and therapeutic compositions capable of protecting animals from parasitic helminth infection and, particularly, from heartworm, infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 08/460,428, filed Jun. 2, 1995, now U.S. Pat. No. 5,912,337,which is a continuation of U.S. application Ser. No. 08/109,391, nowU.S. Pat. No. 5,639,876 which is a continuation-in-part of U.S. patentapplication Ser. No. 08/003,257, filed Jan. 12, 1993, now abandoned, ofU.S. patent application Ser. No. 08/003,389, filed Jan. 12, 1993, nowabandoned; and of U.S. patent application Ser. No. 07/654,226, filedFeb. 12, 1991, now abandoned. Each of these applications is incorporatedby reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to novel parasitic helminth proteins,nucleic acid sequences encoding such proteins and antibodies raisedagainst such proteins. The present invention also includes a method toobtain such nucleic acid sequences and proteins, and a method of usingsuch nucleic acid sequences, antibodies, and proteins to protect animalsfrom infection. The present invention particularly relates to specificDirofilaria immitis nucleic acid sequences and proteins as well as theiruse to protect animals from heartworm infection.

BACKGROUND OF THE INVENTION

Parasitic helminth infections in animals, including humans, aretypically treated by chemical drugs, because there are essentially noefficacious vaccines available. One disadvantage with chemical drugs isthat they must be administered often. For example, dogs susceptible toheartworm are typically treated monthly to maintain protective druglevels. Repeated administration of drugs to treat parasitic helminthinfections, however, often leads to the development of resistant strainsthat no longer respond to treatment. Furthermore, many of the chemicaldrugs are harmful to the animals being treated, and as larger dosesbecome required due to the build up of resistance, the side effectsbecome even greater.

It is particularly difficult to develop vaccines against parasitichelminth infections both because of the complexity of the parasite'slife cycle and because, while administration of parasites or parasiteantigens can lead to the production of a significant antibody response,the immune response is typically not sufficient to protect the animalagainst infection.

As for most parasites, the life cycle of Dirofilaria immitis, thehelminth that causes heartworm, includes a variety of life forms, eachof which presents different targets, and challenges, for immunization.Adult forms of the parasite are quite large and preferentially inhabitthe heart and pulmonary arteries of an animal. Males worms are typicallyabout 12 cm (centimeters) to about 20 cm long and about 0.7 mm to about0.9 mm wide; female worms are about 25 cm to about 31 cm long and about1.0 to about 1.3 mm wide. Sexually mature adults, after mating, producemicrofilariae which are only about 300 μm (micrometers) long and about 7μm wide. The microfilariae traverse capillary beds and circulate in thevascular system of the dog in concentrations of about 10³ to about 10⁵microfilariae per ml of blood. One method of demonstrating infection inthe dog is to detect the circulating microfilariae.

If the dog is maintained in an insect-free environment, the life cycleof the parasite cannot progress. However, when microfilariae areingested by the female mosquito during blood feeding on an infected dog,subsequent development of the microfilariae into larvae occurs in themosquito. The microfilariae go through two larval stages (L1 and L2) andfinally become mature third stage larvae (L3) of about 1.1 mm length,which can then be transmitted back to the dog through the bite of themosquito. It is this L3 stage, therefore, that accounts for the initialinfection. As early as three days after infection, the L3 molt to thefourth larval (L4) stage, and subsequently to the fifth stage, orimmature adults. The immature adults migrate to the heart and pulmonaryarteries, where they mature and reproduce, thus producing themicrofilariae in the blood. “Occult” infection with heartworm in dogs isdefined as that wherein no microfilariae can be detected, but theexistence of the adult heartworms can be determined through thoracicexamination.

Heartworm not only is a major problem in dogs, which typically cannoteven develop immunity upon infection (i.e., dogs can become reinfectedeven after being cured by chemotherapy), but is also becomingincreasingly widespread in other companion animals, such as cats andferrets. Heartworm infections have also been reported in humans. Otherparasitic helminthic infections are also widespread, and all requirebetter treatment, including a preventative vaccine program.

Although many investigators have tried to develop vaccines based onspecific antigens, it is well understood that the ability of an antigento stimulate antibody production does not necessarily correlate with theability of the antigen to stimulate an immune response capable ofprotecting an animal from infection, particularly in the case ofparasitic helminths. A large number of materials are immunogenic andproduce sera which test positive in immunoassays for ability to reactwith the immunizing antigen, but which fail to protect the hosts againstinfection. Antibodies which neutralize the infective agent in in vitroassays are much more likely to protect against challenge in vivo.Accordingly, the use of serum simply resulting from immunization or frominfection by a parasitic helminth to screen for candidate vaccines doesnot provide sufficient specificity to identify protective immunogens. Onthe other hand, serum or other components of blood from immunizedanimals which is demonstrably protective against infection would containantibodies, cells, or other factors that could selectively bind topotential antigens that, if used as therapeutic compositions, wouldelicit immune responses that protect against challenge.

In most infectious diseases, particularly those such as parasiticinfections that have long and complex development courses, it isdifficult to verify the protective effect of serum or T-cells fromexposed animals for use as a screening reagent. First, verification ofprotection against challenge is tedious, since the host animal wouldfirst have to be challenged with the infectious agent and shown to beprotected before it could be shown that antibody components of serum,for example, could be used as a screen. The definition of protectionunder such a regimen is often complex. Second, even if a protectiveeffect against challenge is shown, it is not clear to what components ofthe immune system the protection is due. The protective effect could bedue to antibodies, cells, mediators of the immune system or tocombinations thereof. Thus, although this method of obtaining thescreening reagent is sometimes used, it is time-consuming and does notpermit identification of protective components.

A method to determine the effectiveness of in vivo immunizationprotocols includes implanting diffusion chambers containing infectiousagents into immunized animals and determining the effects of suchimmunizations on the implanted infectious agents. Grieve, et al., 1988,Am. J. Trop. Med. Hyg. 39, p. 373-379, for example, report that dogswhich had been immunized against Dirofilaria immitis infection weresupplied diffusion chambers containing infective larvae. The larvae inthe chambers could then be evaluated for the effect of the previousimmunizations. Abraham, et al., 1988 J. Parasit. 74, p. 275-282, reportthat mice which had been immunized with L3, were supplied diffusionchambers containing D. immitis third-stage larvae, and the effects onthese larvae were used to determine the possible immunity of the miceputatively developed by such immunization. Thus, the papers disclosethat implantation of diffusion chambers containing the infectious agentinto an immunized animal provides a convenient assessment of theeffectiveness of certain directly administered active immunizationprotocols, but do not describe the use of such chambers to monitorpassively transferred protective effects of selected fractions of atarget host bloodstream.

Protection against parasitic helminth infections is difficult to achievebecause, as heretofore stated, the complexity of the parasitic infectionmakes the choice of a candidate immunogen for vaccination verydifficult. Even naturally conferred immunity cannot be assured to exist,as dogs with previous or existing infections with D. immitis can bereinfected (see, for example, Grieve et al., 1983, Epidemiologic Reviews5, p. 220-246). However, this review also reports that there is someevidence of a naturally occurring protective immune response, whichapparently limits the population of mature worms in infected dogs.

Furthermore, it has been possible to induce protective immunityartificially. Wong, et al., 1974, Exp. Parasitol. 35, p. 465-474,reported the immunization of dogs with radiation-attenuated infectivelarvae. The dogs were protected to varying degrees upon challenge.Blair, et al., 1982 in Fifth International Congress of Parasitology,Toronto, Canada, reported successful immunization by infecting the dogsand terminating the infection at the fourth larval stage bychemotherapy.

Grieve, 1989, Proc. Heartworm Symp., p. 187-190, reviewed the status ofattempts to produce vaccines against heartworm in dogs. This reportsummarizes the use of infective larvae implanted in an inert diffusionchamber which permits the influx of cells and/or serum from the host andoutflow of parasite material from the chamber to assess theeffectiveness of inoculation protocols in both dogs and mice. The use ofimmunization with infective larvae was demonstrated to be partiallyeffective in protection against subsequent challenge.

An alternative approach to finding, for example, a heartworm vaccine hasbeen to attempt to identify prominent antigens in the infective stage ofD. immitis. Philipp, et al., 1986, J. Immunol. 136, p. 2621-2627,reports a 35-kilodalton (kD) major surface antigen of D. immitis thirdstage larvae which was capable of immunoprecipitation with sera fromdogs carrying an occult experimental D. immitis infection or with serafrom dogs immunized by irradiated third stage larvae. In addition, thisgroup reported (Davis, et al., 1988, Abstract 404, 37th Annual Meeting,Am. Soc. Trop. Med. Hyg.) three major surface proteins of the L4 havingmolecular weights of 150 kD, 52 kD, and 25 kD. The 25 kD molecule seemedunique to L4 larvae.

Ibrahim, et al., 1989, Parasitol. 99, p. 89-97, using D. immitis L3larvae labeled with ¹²⁵I, showed that a 35 kD and 6 kD component wereshed into the culture medium by developing parasites. They furthershowed that antibodies from immunized rabbits and infected dogsimmunoprecipitated the 35 kD, but not the 6 kD, component.

Scott, et al, 1990, Acta Tropica 47, p. 339-353, reportedcharacterization of the surface-associated molecules of D. immitis L2,L3, and L4 by radiolabeling techniques and SDS-PAGE. They found majorlabeled components of 35 kD and 6 kD in extracts from iodine-labeled L2and L3; lactoperoxidase-catalyzed labeling revealed components ofapparent molecular weights 66 kD, 48 kD, 25 kD, 16.5 kD, and 12 kD.Iodine labeling of surface-associated molecules of L4 gave molecules ofapparent molecular weights of 57 kD, 40 kD, 25 kD, 12 kD, and 10 kD;lactoperoxidase-catalyzed labeling showed additional bands of 45 kD, 43kD, and 3 kD. However, these antigens were identified usinguncharacterized serum sources.

Other approaches to obtaining vaccines against parasites in general havefocused on the production of neutralizing antibodies. For example, bothin vitro studies by Tanner, et al., 1981, Trans. Roy. Soc. Trop. Med.Hyg. 75, p. 173-174 and by Sim et al., 1982, Trans. Roy. Soc. Trop. Med.Hyg. 76, p. 362-370, and in vivo studies by Parab et al., 1988, Immunol.64, p. 169-174, have demonstrated that antibodies are effective alone orwith other immune components in killing filarial L3 from Dipetalonema(Acanthocheilonema) viteae or Brugia malayi. Furthermore, passiveimmunity to Schistosoma mansoni has been transferred from immune rats orhumans to normal mice (see, for example, Sher, et al., 1975, Parasitol.,70, p. 347-357; Jwo et al., 1989, Am. J. Trop. Med. Hyg. 41, p.553-562). None of these studies involved the use of an in vivo assay todetermine the ability of serum, or cellular, components to be a usefulscreening tool for identifying protective antigens. Neither has any ofthese studies yet identified an effective vaccine.

SUMMARY OF THE INVENTION

The present invention includes an isolated parasitic helminth nucleicacid sequence capable of hybridizing, under stringent conditions, to atleast a portion of D. immitis nucleic acid sequence p4 and/or to atleast a portion of D. immitis nucleic acid sequence p22U. A preferredisolated nucleic acid sequence-encodes a protein capable of selectivelybinding to at least one component of immune serum that is capable ofinhibiting helminth development. Another preferred nucleic acid sequenceincludes an oligonucleotide capable of hybridizing to at least one ofthe D. immitis nucleic acid sequences under stringent hybridizationconditions. The present invention also includes recombinant moleculesand recombinant cells that include isolated nucleic acids of the presentinvention. Also included is a method to produce isolated nucleic acidsequences of the present invention.

Another embodiment of the present invention includes an isolatedparasitic helminth protein, or mimetope, thereof, capable of selectivelybinding to at least one component of immune serum that is capable ofinhibiting helminth development, the protein being encoded by aparasitic helminth nucleic acid sequence capable of hybridizing, understringent conditions, to at least a portion of D. immitis nucleic acidsequence p4 and/or to at least a portion of D. immitis nucleic acidsequence p22U. Preferred immune serum is derived from an animal that isimmune to infection by the helminth, and preferably from an animalimmunized with third stage and/or fourth stage larvae. Also included isa method to produce such isolated proteins and mimetopes of the presentinvention.

Yet another embodiment of the present invention is an antibody capableof selectively binding to a parasitic helminth protein or mimetopethereof, the antibody being produced by a method that includesadministering to an animal an effective amount of an isolated protein ormimetope of the present invention. Also included is a method to producesuch antibodies.

Yet another embodiment of the present invention is a therapeuticcomposition capable of protecting an animal from parasitic helminthinfection when administered to the animal in an effective manner. Thetherapeutic composition includes at least one of the followingtherapeutic compounds: an isolated nucleic acid sequence of the presentinvention, an isolated protein or mimetope of the present invention,and/or an antibody of the present invention. The composition can alsoinclude an excipient, adjuvant, and/or carrier. Preferably, thetherapeutic composition protects the animal against heartworm. Thepresent invention also includes a method to protect an animal fromparasitic helminth infection by administering such therapeuticcompositions.

Yet another embodiment of the present invention includes a therapeuticcomposition capable of protecting an animal from parasitic helminthinfection when administered to the animal in an effective manner, thecomposition including a compound capable of substantially interferingwith the function of a parasitic helminth LDL receptor-related proteinclass A cysteine-rich motif. A preferred therapeutic composition is aprotein encoded by an isolated nucleic acid sequence capable ofhybridizing, under stringent conditions, to at least a portion ofDirofilaria immitis nucleic acid sequence p4. The present invention alsoincludes a method to protect animals from parasitic helminth infectionusing such therapeutic compositions.

Preferred parasitic helminths of the present invention includenematodes, cestodes and trematodes, with filarial, ascarid, strongyleand trichostrongyle nematodes being more preferred. Dirofilaria,Onchocerca, Brugia, Wuchereria, Loa, Acanthocheilonema, Dipetalonema,Setaria, Parafilaria and Stephanofilaria filarial nematodes are evenmore preferred, and Dirofilaria immitis, the parasite that causesheartworm, is even more preferred.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a chromatogram of the separation of larval ES by cationexchange chromatography.

FIG. 2 depicts a chromatogram of the separation of tryptic fragments ofP22U by C₁₈ reverse phase chromatography; P22U was purified by cationexchange and C₄ reverse phase chromatography.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes isolated parasitic helminth proteins andmimetopes thereof that are capable of selectively binding to at leastone component of a serum collected from an animal that is immune toinfection by the helminth, the serum being capable of inhibitinghelminth development. The ability of such proteins and mimetopes toselectively bind to components in such a serum is believed to suggestthe ability of such proteins and mimetopes to protect an animal fromparasite infection when such proteins and/or mimetopes are administeredto an animal in an effective manner.

Animals that are immune to infection by parasitic helminths are animalsthat exhibit an immune response that is sufficient to protect the animalfrom such infection. Immune animals typically are animals that have beenadministered larval, adult and/or microfilarial helminths in a mannereffective to elicit a protective response, preferably using irradiatedhelminths or a chemically-abbreviated infection protocol. For example,dogs receiving chemically abbreviated D. immitis larval infectionsexhibit significant immunity to challenge infections. Furthermore, seraobtained from such dogs are effective in passively transferring larvalkilling and stunting capabilities to mice. Preferred immune animals arethose that have been immunized against helminth larvae, particularlyagainst L3 and/or L4 larvae, since, in accordance with the presentinvention, it is particularly desirable to prevent L3 larvae introducedinto an animal from developing into adult parasites. It should be noted,however, that immune animals do not preclude naturally-infected animalsthat generate protective antibodies.

According to the present invention, an isolated, or biologically pure,parasitic helminth protein, is a protein that has been removed from itsnatural milieu. As such, “isolated” and “biologically pure” do notnecessarily. reflect the extent to which the protein has been purified.An isolated parasitic helminth protein can be obtained from its naturalsource. Alternatively, the isolated parasitic helminth protein can beproduced using recombinant DNA technology or chemical synthesis.Isolated proteins include full-length proteins as well as modifiedversions of the protein in which amino acids have been deleted (e.g., atruncated version of the protein, such as a peptide), inserted,inverted, substituted and/or derivatized (e.g., glycosylated,phosphorylated, acetylated) such that the modified version of theprotein has a biological function substantially similar to that of thenatural protein (i.e., functionally equivalent to the natural protein).Modifications can be accomplished by techniques known in the artincluding, but not limited to, direct modifications to the protein ormodifications to the gene encoding the protein using, for example,classic or recombinant DNA techniques to effect random or targetedmutagenesis. Isolated proteins of the present invention, includingmodified versions thereof, can be identified in a straight-forwardmanner by the proteins' ability to selectively bind to at least onecomponent of anti-parasitic helminth immune serum. As used herein,immune serum refers to serum that is capable of inhibiting helminthdevelopment that preferably is derived (e.g., obtained from) an animalthat is immune to the helminth. The minimum size of isolated proteins ofthe present invention is sufficient to form an epitope, a size that istypically at least about 7 to about 9 amino acids. As is appreciated bythose skilled in the art, an epitope can include amino acids thatnaturally are contiguous to each other as well as amino acids that, dueto the tertiary structure of the natural protein, are in sufficientlyclose proximity to form an epitope.

In accordance with the present invention, a mimetope refers to anycompound that is able to mimic the ability of an isolated protein of thepresent invention to selectively bind to at least one component ofanti-parasitic helminth immune serum. A mimetope can be a peptide thathas been modified to decrease its susceptibility to degradation but thatstill retains its selective binding ability. Other examples of mimetopesinclude, but are not limited to, anti-idiotypic antibodies, or fragmentsthereof, that include at least one binding site that mimics one or moreepitopes of an isolated protein; non-proteinaceous immunogenic portionsof an isolated protein (e.g., carbohydrate structures); and synthetic ornatural organic molecules, including nucleic acids, that have astructure similar to at least one epitope of an isolated protein of thepresent invention. Such mimetopes can be obtained, for example, byaffinity chromatography techniques using immune sera of the presentinvention or antibodies raised against isolated proteins of the presentinvention.

As used herein, the term “selectively binds to” refers to the ability ofisolated proteins and mimetopes thereof to bind to serum collected fromanimals that have been exposed to parasitic helminths (either throughnatural infection or through administration of helminths) butessentially not to bind, according to standard detection techniques(such as those described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989, which isincorporated herein by reference in its entirety) to serum collectedfrom animals that have not been exposed to parasitic helminths (i.e.,naive animals). Preferably, the isolated proteins and mimetopes are ableto bind to anti-parasitic helminth immune serum with high affinity. Theability of a protein or mimetope thereof to selectively bind toanti-parasitic helminth immune serum can be measured using a variety ofmethods known to those skilled in the art including immunoblot assays,immunoprecipitation assays, enzyme immunoassays (e.g., ELISA),radioimmunoassays, immunofluorescent antibody assays and immunoelectronmicroscopy. It should be noted that the ability of an isolated proteinor mimetope thereof to selectively bind to immune serum raised against acertain stage of helminth development does not preclude the isolatedprotein or mimetope from being able to also bind to immune serum raisedagainst other stages of helminth development. For example, the abilityof an isolated protein or mimetope thereof to selectively bind to ananti-larval immune serum does not preclude the isolated protein ormimetope from being able to also bind to anti-microfilarial and/oranti-adult immune serum.

One embodiment of the present invention is the use of anti-parasitichelminth immune serum to identify isolated proteins and mimetopes of thepresent invention, a technique referred to herein as immune serumscreening assay. Immune serum can be raised against a parasitic helminthby administering the helminth to an animal under conditions that elicitan immune response. Immune serum can be raised against larval,microfilarial, and/or adult helminths, preferably against larvae, andmore preferably against L3 and/or L4 larvae. Immune sera of the presentinvention are capable not only of inhibiting development of the speciesof helminth that elicited the immune response, but also of helminthspecies that immunologically cross-react with the immune sera. Due tothe similarity between helminths, immune sera of the present inventionare capable of reacting with a large variety of helminths. Inhibitingthe development of helminths includes killing, reducing the growth of,blocking the maturation of, altering the morphology of, altering themetabolism of, and/or otherwise being detrimental to the helminth.

Any animal that is capable of mounting an immune response to protectitself from helminth infection is a suitable animal to which helminthscan be administered and from which immune serum can be collected. Forexample, a preferred animal from which to collect serum capable ofinhibiting the development of Dirofilaria immitis is a dog that has beenadministered L3 and/or L4 Dirofilaria immitis larvae under conditionsthat elicit an immune response.

The ability of immune serum of the present invention to inhibitparasitic helminth development can be determined in a number of ways. Apreferred method to monitor the ability of immune serum to inhibit thedevelopment of an infectious agent is disclosed in U.S. patentapplication Ser. No. 07/654,226, referenced above. As disclosed therein,for example, the ability of, an anti-parasitic helminth larval immuneserum to inhibit larval development can be determined as follows.Briefly, a naive animal (i.e., an animal not previously exposed toparasitic helminth larvae) is implanted with at least one diffusionchamber containing helminth larvae, preferably L3 larvae. The animal isalso administered either the anti-larval immune serum to be tested or acontrol non-immune serum, preferably at a site near the diffusionchambers. After a suitable period of time, for example, from about threeto about four weeks for Dirofilaria immitis larvae implanted in mice,the diffusion chambers are removed, and the effects of the immune serumon larval growth and development are determined by, for example,comparing larval growth and survival in chambers exposed to anti-larvalimmune serum with the growth and survival of larvae in diffusionchambers exposed to non-immune serum. A significant number of larvaeexposed to anti-larval immune serum are either killed or stuntedcompared to larvae exposed to non-immune serum.

U.S. patent application Ser. No. 07/654,226 further discloses use of theimmune serum screening assay to screen for, and hence identify, desiredproteins that selectively bind to the immune serum. Briefly, the immuneserum can be contacted with a protein-containing composition underconditions that permit selective binding by desired proteins tocomponents in the serum. Complexes between the proteins and serumcomponents are recovered, the proteins are separated from the serumcomponents and are then analyzed. Nucleic acid sequence encoding suchproteins can be identified using known recombinant DNA techniques, suchas those described in Sambrook et al., ibid. In another embodiment, theimmune serum screening assay can be used to identify nucleic acidsequences encoding isolated proteins of the present invention byscreening parasite helminth expression cDNA libraries with immune seraof the present invention to identify proteins expressed by individualclones that are capable of selectively binding to the immune sera. Theimmune serum screening assay can also be used to identify mimetopescapable of selectively binding to immune serum, such as to anti-L3and/or L4 larval immune serum. Mimetopes can also be designed orimproved using information derived from proteins identified by theimmune serum screening assay. It should be appreciated that not onlyserum, but also other immunogenic components of bodily fluids collectedfrom animals immune to helminth infection, such as cells, specificantibodies, and fragments thereof, can be used in the immune serumscreening assay.

As disclosed in U.S. patent application Ser. No. 07/654,226, anti-larvalimmune serum has been used to identify nematode proteins expressedduring L3 and/or L4 that have molecular weights of 66 kD, 65 kD, 59 kD,39 kD, 33 kD, 23/24 kD, 22/20.5 kD and 14 kD, as determined by theirmigration patterns when submitted to Tris-glycine SDS PAGE. Nucleic acidsequences encoding these proteins can be identified using anti-L3 and/orL4 larval immune serum to screen larval nematode cDNA expressionlibraries. U.S. application Ser. No. 08/003,257, referenced above,discloses 22-kD and 20.5-kD nematode proteins (sizes determined by Trisglycine SDS-PAGE), referred to herein as P22L and P20.5, and nucleicacid sequences that encode them, herein referred to as p22L and p20.5.P22L and P20.5 are the same protein except that P22L has a hydrophobicleader sequence attached to the P20.5 protein. U.S. application Ser. No.08/003,389, referenced above, discloses 39-kD nematode proteins (sizesdetermined by Tris glycine SDS-PAGE), referred to herein as P39, andnucleic acid sequences that encode them, herein referred to as p39. Thepresent application, by incorporating U.S. application Ser. Nos.07/654,226, 08/003,257 and 08/003,389 by reference herein in theirentireties, includes D. immitis P22L, D. immitis P20.5, D. immitis P39,additional parasitic helminth proteins sharing significant homology withD. immitis P22L, D. immitis P20.5, or D. immitis P39, nucleic acidsequences encoding any of these proteins, mimetopes of any of theseproteins, and antibodies that selectively bind to any of these proteins,as well as uses of these proteins, mimetopes, nucleic acid sequences,and antibodies.

One embodiment of the present invention is an isolated parasitichelminth nucleic acid sequence that encodes an isolated protein of thepresent invention. As used herein, an isolated parasitic helminthnucleic acid sequence is a nucleic acid sequence that has been removedfrom its natural milieu. As such, “isolated” does not reflect the extentto which the nucleic acid sequence has been purified. An isolatednucleic acid sequence can be DNA, RNA, or derivatives of either DNA orRNA. Isolated nucleic acid sequences of the present invention includesequences that encode at least one epitope capable of selectivelybinding to immune sera of the present invention as well asoligonucleotides that can function in a variety of ways, including, butnot limited to, as probes, primers, and therapeutic agents using, forexample, antisense, triplex formation- and/or ribozyme-basedtechnologies. An isolated parasitic helminth nucleic acid sequence canbe obtained from its natural source either as an entire gene or aportion thereof, the minimal size of a portion being a size that canform a stable hybrid with a similar nucleic acid sequence understringent conditions. As such, isolated nucleic acid sequences caninclude regulatory regions that control expression of the correspondingcoding region (e.g., transcription or translation control regions),full-length or partial coding regions, and combinations thereof.Isolated parasitic helminth nucleic acid sequences can also be producedusing recombinant DNA technology (e.g., PCR amplification, cloning) orchemical synthesis. Isolated parasitic helminth nucleic acid sequencesinclude functional equivalents of natural sequences, including, but notlimited to, natural allelic variants and modified nucleic acid sequencesin which nucleotides have been inserted, deleted, substituted, and/orinverted in such a manner that such modifications do not substantiallyinterfere with the nucleic acid sequence's ability to encode an epitoperecognized by immune sera of the present invention or do notsubstantially interfere with the ability of the nucleic acid sequence toform stable hybrids under stringent conditions with natural isolates. Asused herein, stringent hybridization conditions refer to standardhybridization conditions under which nucleic acid sequences, includingoligonucleotides, are used to identify similar sequences. Such standardconditions are disclosed, for example, in Sambrook et al., ibid.Examples of such conditions are provided in the Examples section.

Functionally equivalent nucleic acid sequences can be obtained usingmethods known to those skilled in the art (see, for example, Sambrook etal., ibid.). For example, nucleic acid sequences can be modified using avariety of techniques including, but not limited to, classic mutagenesistechniques and recombinant DNA techniques, such as site-directedmutagenesis, chemical treatment of a nucleic acid to induce mutations,restriction enzyme cleavage of a nucleic acid fragment, ligation ofnucleic acid fragments, polymerase chain reaction (PCR) amplificationand/or mutagenesis of selected regions of a nucleic acid sequence,synthesis of oligonucleotide mixtures and ligation of mixture groups to“build” a mixture of nucleic acid sequences, and combinations thereof.Functionally equivalent nucleic acids can be selected from a mixture ofmodified nucleic acid sequences by screening for the function of theprotein encoded by the nucleic acid sequence (e.g., ability to bind toimmune serum) and/or by hybridization with natural nucleic acidsequences under stringent conditions.

Due to the similarity between parasitic helminth genomes, isolatedproteins and corresponding nucleic acid sequences of the presentinvention can be from any parasitic helminth. Preferred helminthsinclude nematode, cestode and trematode parasites. More preferredhelminths include filarial, ascarid, strongyle and trichostrongylenematodes. Even more preferred helminths include Dirofilaria,Onchocerca, Brugia, Wuchereria, Loa, Acanthocheilonema, Dipetalonema,Setaria, Parafilaria and Stephanofilaria filarial nematodes. Aparticularly preferred parasitic helminth of the present invention isDirofilaria immitis, the filarial nematode that causes heartworm.

One embodiment of the present invention is an isolated parasitichelminth nucleic acid sequence that is capable of hybridizing, understringent conditions, to at least a portion of D. immitis nucleic acidsequence p4. A protein encoded by such a nucleic acid sequence ispreferably capable of selectively binding to at least one component ofanti-parasitic helminth immune serum, and more preferably to anti-L3and/or L4 larval immune serum. D. immitis nucleic acid sequence p4, alsoreferred to as D. immitis p4, is a nucleic acid sequence of about 913nucleotides in length that has been isolated from a D. immitis L3 and/orL4 cDNA, expression library using immune serum collected from a dog thatwas immunized by repeated chemically abbreviated infections (e.g.,infect with about 200 L3, wait about 60 days, treat with ivermectin,wait about 60 days, reinfect, etc.). Sequencing of D. immitis p4 hasresulted in the nucleic acid sequence disclosed in SEQ ID NO:1. Itshould be noted that sequencing technology is not entirely error-freeand that SEQ ID NO:1, as such, represents an apparent nucleic acidsequence of D. immitis p4. The deduced translation of SEQ ID NO:1,represented in SEQ ID NO:2, suggests that D. immitis p4 comprises anopen reading frame of about 303 amino acids and, as such, representsonly a portion of the entire coding sequence of the gene. The nucleicacid contained in D. immitis p4, however, is sufficient to encode aprotein that selectively binds with anti-D. immitis larval immune serum,as demonstrated by the manner in which the nucleic acid sequence wasisolated. The deduced translation of SEQ ID NO:1 suggests that theprotein encoded by D. immitis p4 has a molecular weight of about 35.5kilodaltons (kD) and an estimated pI of 4.26.

The protein encoded by D. immitis p4 is further characterized by havingan LDL receptor-related protein (LDLr) class A cysteine-rich motif ofabout 9 amino acids that is also found in several other proteins,including mammalian low density lipoprotein (LDL) receptors, LDLreceptor-related proteins, human and mouse alpha-2-macroglobulinreceptors and rat renal GP 330 glycoprotein. Each of these proteins,including D. immitis P4, share the sequence DDCGDGSDE (i.e., AsparticAcid—Aspartic Acid—Cysteine—Glycine—AsparticAcid—Glycine—Serine—Aspartic Acid—Glutamic Acid), denoted herein as SEQID NO:5. A conserved stretch of eight of the nine amino acids is alsofound in the free-living (i.e., non-parasitic) nematode Caenorhabditiselegans LDL receptor-related protein and C. elegans basement membraneproteoglycan. This LDLr class A, cysteine-rich motif is likely to beconserved in proteins encoded by p4-related sequences of other helminths(i.e., nucleic acid sequences that hybridize under stringent conditionswith D. immitis p4). As such, p4-related nucleic acid sequences may beidentified using oligonucleotide probes that encode such LDLr class Amotifs. Furthermore, the LDLr class A motif in P4-related proteinsrepresents a target for development of therapeutic compositions toprotect animals from parasitic helminth infection, as discussed below.

The present invention includes nucleic acid sequences from any parasitichelminth that hybridize under stringent conditions to at least a portionof D. immitis nucleic acid sequence p4, the minimal size of the portionbeing defined by the hybridization conditions. Due to the similaritiesbetween parasitic helminths, as heretofore disclosed, one can use D.immitis p4 sequences to obtain other parasitic helminth nucleic acidsequences that are capable of hybridizing, under stringent conditions,to at least a portion of D. immitis p4. Preferred helminths areheretofore disclosed.

Particularly preferred nucleic acid sequences of the present inventioninclude D. immitis nucleic acid sequence p4, nucleic acid sequencesincluding D. immitis p4, and nucleic acid sequence comprising fragmentsof D. immitis p4 (including functional equivalents of any of thesenucleic acid sequences). Knowing the sequence of D. immitis p4 allowsone skilled in the art to make copies of the sequence as well as toobtain nucleic acid sequences including D. immitis p4 and nucleic acidsequences that contain fragments of D. immitis p4. As such, particularlypreferred isolated nucleic acid sequences include SEQ ID NO:1 or afunctional equivalent thereof, a nucleic acid sequence containing atleast a portion of SEQ ID NO:1 or a functional equivalent thereof, and afragment of SEQ ID NO:1 or a functional equivalent thereof, assuming theaccuracy of SEQ ID NO:1.

The present invention also includes an isolated parasitic helminthprotein that is encoded, at least in part, by a parasitic helminthnucleic acid sequence capable of hybridizing, under stringentconditions, to at least a portion of D. immitis nucleic acid sequencep4, as well as mimetopes of such a protein. Preferably, the protein ormimetope thereof is also capable of selectively binding to at least onecomponent of anti-parasitic helminth immune serum, and more preferablyto anti-L3 and/or L4 larval immune serum. Preferred isolated parasitichelminth proteins or mimetopes thereof are capable of protecting ananimal from helminth infection when administered to the animal in aneffective manner. Preferred helminths are heretofore disclosed.Particularly preferred isolated proteins include proteins encoded byDirofilaria immitis nucleic acid sequence p4, a nucleic acid sequenceincluding D. immitis p4, or a nucleic acid sequence comprising afragment of D. immitis p4. As such, particularly preferred isolatedproteins are those encoded by SEQ ID NO:1 or a functional equivalentthereof, a nucleic acid sequence containing at least a portion of SEQ IDNO:1 or a functional equivalent thereof, and a fragment of SEQ ID NO:1or a functional equivalent thereof, as well as proteins that contain atleast a portion of SEQ ID NO:2, assuming the accuracy of SEQ ID NO:1 andSEQ ID NO:2.

One embodiment of the present invention is an isolated parasitichelminth nucleic acid sequence that is capable of hybridizing, understringent conditions, to at least a portion of D. immitis nucleic acidsequence p22U. A protein encoded by such a nucleic acid sequence ispreferably capable of selectively binding to at least one component ofanti-parasitic helminth immune serum, and more preferably to anti-L3and/or L4 larval immune serum. D. immitis nucleic acid sequence p22U,also referred to as D. immitis p22U, encodes at least a substantialportion of a basic D. immitis protein, referred to as D. immitis P22Uprotein, that migrates at an apparent molecular weight of about 22 kDwhen submitted to Tris-glycine SDS (sodium dodecyl sulfate) PAGE(polyacrylamide gel electrophoresis). D. immitis P22U protein has beenidentified in larval ES (excretory-secretory) extracts as well as inextracts of L3, L4 and adults. D. immitis p22U is about 1016 nucleotidesin length. Sequencing of D. immitis p22U has resulted in the nucleicacid sequence disclosed in SEQ ID NO:3. It should be noted thatsequencing technology is not entirely error-free and that SEQ ID NO:3,as such, represents an apparent nucleic acid sequence of D. immitisp22U. The deduced translation of SEQ ID NO:3, represented in SEQ IDNO:4, suggests that D. immitis p22U includes an open reading frame ofabout 208 amino acids followed by a stop codon. The translation startsite is as yet unknown although there are two “in-frame” potential startcodons at about amino acid 13 and about amino acid 19 of corresponding(i.e., deduced) amino acid sequence SEQ ID NO:4. The deduced amino acidsequence suggests a protein having a molecular weight of about 22 kD andan estimated pI of about 9.6.

D. immitis p22U can be isolated in a number of ways including, but notlimited to, screening an L3, L4, or adult expression cDNA library withappropriate immune serum or with antibodies raised against D. immitisP22U protein. Alternatively, amino acid sequence information can bederived from purified D. immitis P22U protein that can be used to designoligonucleotide probes and/or primers that can be used to screen and/oramplify sequences from an appropriate cDNA or genomic library.

The present invention includes nucleic acid sequences from any parasitichelminth that hybridize under stringent conditions to at least a portionof D. immitis nucleic acid sequence p22U, the minimal size of theportion being defined by the hybridization conditions. Due to thesimilarities between parasitic helminths, as heretofore disclosed, onecan use D. immitis p22U to obtain other parasitic helminth nucleic acidsequences that are capable of hybridizing, under stringent conditions,to at least a portion of D. immitis p22U. Preferred helminths areheretofore disclosed.

Particularly preferred p22U-related nucleic acid sequences of thepresent invention include Dirofilaria immitis nucleic acid sequencep22U, nucleic acid sequences including D. immitis p22U, and nucleic acidsequence comprising fragments of D. immitis p22U (including functionalequivalents of each of these nucleic acid sequences). Knowing thesequence of D. immitis p22U allows one skilled in the art to make copiesof the sequence as well as to obtain nucleic acid sequences including D.immitis p22U and nucleic acid sequences that contain fragments of D.immitis p22U. As such, particularly preferred isolated nucleic acidsequences include SEQ ID NO:3 or a functional equivalent thereof, anucleic acid sequence containing at least a portion of SEQ ID NO:3 or afunctional equivalent thereof, and a fragment of SEQ ID NO:3 or afunctional equivalent thereof, assuming the accuracy of SEQ ID NO:3.

The present invention also includes an isolated parasitic helminthprotein that is encoded, at least in part, by a parasitic helminthnucleic acid sequence capable of hybridizing, under stringentconditions, to at least a portion of D. immitis nucleic acid sequencep22U, as well as mimetopes of such a protein. Preferably, the protein ormimetope thereof is also capable of selectively binding to at least onecomponent of anti-parasitic helminth immune serum, and more preferablyto anti-L3 and/or L4 larval immune serum. Preferred isolated parasitichelminth proteins or mimetopes thereof are capable of protecting ananimal from helminth infection when administered to the animal in aneffective manner. Preferred helminths are heretofore disclosed.Particularly preferred isolated proteins include proteins encoded byDirofilaria immitis nucleic acid sequence p22U, a nucleic acid sequenceincluding D. immitis p22U, or a nucleic acid sequence comprising afragment of D. immitis p22U. As such, particularly preferred isolatedproteins are those encoded by SEQ ID NO:3 or a functional equivalentthereof, a nucleic acid sequence containing at least a portion of SEQ IDNO:3 or a functional equivalent thereof, and a fragment of SEQ ID NO:3or a functional equivalent thereof, as well as proteins that contain atleast a portion of SEQ ID NO:4, assuming the accuracy of SEQ ID NO:3 andSEQ ID NO:4.

Isolated nucleic acid sequences of the present invention can alsoinclude a nucleic acid that is capable of hybridizing, under stringentconditions, to at least a portion of both D. immitis nucleic acidsequence p4 and D. immitis nucleic acid sequence p22U. Such a nucleicacid sequence can encode a protein including portions of both P4 andP22U. Alternatively, such a nucleic acid sequence could encode both aP4-related and a P22-related protein.

The present invention also includes oligonucleotides that are capable ofhybridizing, under stringent conditions, to complementary regions ofother, preferably longer, nucleic acid sequences of the presentinvention, such as to complementary regions of D. immitis nucleic acidsequence p4, complementary regions of nucleic acid sequences thatinclude at least a portion of D. immitis p4, complementary region ofnucleic acid sequences that hybridize under stringent conditions to D.immitis p4, complementary regions of D. immitis nucleic acid sequencep22U, complementary regions of nucleic acid sequences that include atleast a portion of D. immitis p22U, and complementary regions of nucleicacid sequences that hybridize under stringent conditions to D. immitisp22U. The oligonucleotides can be RNA, DNA, or derivatives of either.The minimal size of such oligonucleotides is the size required to form astable hybrid between a given oligonucleotide and the complementarysequence on another nucleic acid sequence of the present invention. Assuch, the size is dependent on nucleic acid composition and percenthomology between the oligonucleotide and complementary sequence as wellas upon hybridization conditions per se (e.g., temperature, saltconcentration). For AT-rich nucleic acid sequences, such as those of D.immitis, oligonucleotides typically are, at least about 15 to about 17bases in length. The size of the oligonucleotide must also be sufficientfor the use of the oligonucleotide in accordance with the presentinvention. Oligonucleotides of the present invention can be used in avariety of applications including, but not limited to, as probes toidentify additional nucleic acid sequences, as primers to amplify orextend nucleic acid sequences, or in therapeutic applications toinhibit, for example, expression of nucleic acid sequences intoparasitic helminth proteins that are important in the life cycle of theparasite. Such therapeutic applications include the use of sucholigonucleotides in, for example, antisense-, triplex formation-, and/orribozyme-based technologies.

Isolated nucleic acid sequences of the present invention, such asnucleic acid sequences that hybridize under stringent conditions witheither D. immitis p4 or D. immitis p22U, can be obtained in a variety ofways. For example, an isolated nucleic acid sequence of the presentinvention can be obtained by a method that includes induction of a L3and/or L4 expression library under conditions that promote production oflarval proteins encoded by the library; contacting the library withimmune serum collected from an animal that is immune to infection by L3and/or L4; and selecting a colony or phage plaque that contains anucleic acid sequence encoding a protein capable of selectively bindingto the serum. Conventional culturing and selection methods are taught,for example, in Sambrook et al., ibid. An example of this methodology isalso provided in the Examples section.

In another embodiment, an isolated nucleic acid sequence is obtained bya method including contacting, under stringent hybridization conditions,at least one oligonucleotide with a parasitic helminth cDNA library,such that the oligonucleotide includes nucleic acid sequences thatencode at least a portion of D. immitis P4 and/or D. immitis P22U; andselecting a colony or phage plaque having a nucleic acid sequence thathybridizes under stringent conditions with the oligonucleotide.Alternatively, oligonucleotide primers, including nucleic acid sequencesthat encode at least portions of D. immitis P4 and/or D. immitis P22U,can be used to amplify, by polymerase chain reaction (PCR)amplification, nucleic acid sequences that include at least a portion ofD. immitis p4 and/or D. immitis p22U. An example of these methodologiesis provided in the Examples section.

In yet another embodiment, an isolated nucleic acid sequence is obtainedby a method including contacting a collection of nucleic acid sequences,such as a parasitic helminth cDNA library, with D. immitis p4 or aportion thereof, or with D. immitis p22U or a portion thereof, understringent hybridization conditions; and identifying a nucleic acidsequence that hybridizes to either D. immitis p4 or the portion thereof,or with D. immitis p22U or the portion thereof, under such conditions.Such a technique can be used to clone a nucleic acid sequence usingstandard hybridization techniques or to amplify a nucleic acid sequenceusing PCR amplification. Alternatively, serum raised against D. immitisP4 or D. immitis P22U could be used to screen cDNA expression libraries.

The present invention also includes recombinant vectors, which include aparasitic helminth nucleic acid sequence of the present inventioninserted into any vector capable of delivering the nucleic acid into ahost cell. The vector contains heterologous nucleic acid sequences, thatis nucleic acid sequences that are not naturally found adjacent toparasitic helminth nucleic acid sequences of the present invention andthat preferably are derived from a species other than the species fromwhich the parasitic helminth nucleic acid sequences are derived. Thevector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid. Recombinant vectors can be used inthe cloning, sequencing, and/or otherwise manipulating of nucleic acidsequences of the present invention. One type of recombinant vector,herein referred to as a recombinant molecule and described in moredetail below, can be used in the expression of nucleic acid sequences ofthe present invention. Preferred recombinant vectors are capable ofreplicating in the transformed cell. Preferred nucleic acid sequences toinclude in recombinant vectors of the present invention includeparasitic helminth nucleic acid sequences capable of hybridizing, understringent conditions, to at least a portion of Dirofilaria immitisnucleic acid sequence p4 or to at least a portion of Dirofilaria immitisnucleic acid sequence p22U. Particularly preferred nucleic acidsequences with which to transform cells include D. immitis nucleic acidsequence p4, nucleic acid sequences including D. immitis p4, nucleicacid sequence comprising fragments of D. immitis p4, D. immitis nucleicacid sequence p22U, nucleic acid sequences including D. immitis p22U,and nucleic acid sequence comprising fragments of D. immitis p22U.

Isolated proteins of the present invention can be produced in a varietyof ways, including production and recovery of natural proteins,production and recovery of recombinant proteins, and chemical synthesis.In one embodiment, an isolated protein of the present invention is goproduced by culturing a cell capable of expressing the protein underconditions effective to produce said protein, and recovering theprotein. A preferred cell to culture is a recombinant cell that iscapable of expressing the protein, the recombinant cell being producedby transforming a host cell with one or more nucleic acid sequences ofthe present invention. Transformation of a nucleic acid sequence into ahost cell can be accomplished by any method by which a nucleic acidsequence can be inserted into a cell. Transformation techniques include,but are not limited to, transfection, electroporation, microinjection,lipofection, adsorption, and protoplast fusion. A recombinant cell mayremain unicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid sequences of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of a host cell in such a manner that their abilityto be expressed is retained. Preferred nucleic acid sequences with whichto transform cells include parasitic helminth nucleic acid sequences.capable of hybridizing, under stringent conditions, to at least aportion of Dirofilaria immitis nucleic acid sequence p4 or to at least aportion of Dirofilaria immitis nucleic acid sequence p22U. Particularlypreferred nucleic acid sequences with which to transform cells includeD. immitis nucleic acid sequence p4, nucleic acid sequences including D.immitis p4, nucleic acid sequence comprising fragments of D. immitis p4,D. immitis nucleic acid sequence p22U, nucleic acid sequences includingD. immitis p22U, and nucleic acid sequence comprising fragments of D.immitis p22U.

Suitable host cells to transform include any cell that can betransformed. Host cells can be either untransformed cells or cells thatare already transformed with at least one nucleic acid sequence. Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing isolated proteins of the presentinvention or can be capable of producing such proteins after beingtransformed with at least one nucleic acid sequence of the presentinvention. Host cells of the present invention can be any cell capableof producing an isolated protein of the present invention, includingbacterial, yeast, other fungal, insect, animal, and plant cells.Preferred host cells include bacterial, mycobacterial, yeast, insect andmammalian cells, and more preferred host cells include Salmonella,Escherichia, Bacillus, Saccharomyces, Spodoptera, Mycobacteria,Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dogkidney cell line for canine herpesvirus cultivation), CRFK cells (normalcat kidney cell line for feline herpesvirus cultivation) and COS cells.Particularly preferred host cells are Escherichia coli, including E.coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium,including attenuated strains such as UK-1 _(x)3987 and SR-11 _(x)4072;Spodoptera frugiperda; Trichoplusia ni; MDCK cells; and CRFK cells.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid sequences of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. A cell can be transformed with one or more recombinantmolecules. The phrase operatively linked refers to insertion of anucleic acid sequence into an expression vector in a manner such thatthe sequence is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell, of replicating within the hostcell, and of effecting expression of a specified nucleic acid sequence.Expression vectors can be either prokaryotic or eukaryotic, and aretypically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in recombinant cells of the present invention, including inbacterial, yeast, other fungal, insect, animal, and plant cells.Preferred expression vectors of the present invention can direct geneexpression in bacterial, yeast, insect and mammalian cells and morepreferably in the cell types heretofore disclosed.

Expression vectors of the present invention may also contain secretorysignals to enable an expressed parasitic helminth protein to be secretedfrom its host cell or may contain fusion sequences which lead to theexpression of inserted nucleic acid sequences of the present inventionas fusion proteins. Eukaryotic recombinant molecules may includeintervening and/or untranslated sequences surrounding and/or withinparasitic helminth nucleic acid sequences.

Nucleic acid sequences of the present invention can be operativelylinked to expression vectors containing regulatory sequences such aspromoters, operators, repressors, enhancers, termination sequences,origins of replication, and other regulatory sequences that arecompatible with the host cell and that control the expression of thenucleic acid sequences. In particular, recombinant molecules of thepresent invention include transcription control sequences. Transcriptioncontrol sequences are sequences which control the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, helminths, helminth cells, insect and mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,bacteriophage lambda (such as lambda p_(L) and lambda p_(R)),bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6,bacteriophage SP01, metallothionein, alpha mating factor, Pichia alcoholoxidase, alphavirus subgenomic promoters (such as Sindbis virussubgenomic promoters) baculovirus, Heliothis zea insect virus, vacciniavirus, adenovirus, simian virus 40, retrovirus actin, heat shock,phosphate and nitrate transcription control sequences as well as othersequences capable of controlling gene expression in prokaryotic oreukaryotic cells. Transcription control sequences of the presentinvention can also include naturally occurring transcription controlsequences previously associated with a nucleic acid sequence prior toisolation.

A recombinant molecule of the present invention can be any nucleic acidsequence heretofore described operatively linked to any transcriptioncontrol sequence capable of effectively regulating expression of thenucleic acid sequence in the cell to be transformed. Preferredrecombinant molecules include parasitic helminth nucleic acid sequencescapable of hybridizing, under stringent conditions, to at least aportion of Dirofilaria immitis nucleic acid sequence p4 or to at least aportion of Dirofilaria immitis nucleic acid sequence p22U. Particularlypreferred recombinant molecules contain D. immitis nucleic acid sequencep4, nucleic acid sequences including D. immitis p4, nucleic acidsequence comprising fragments of D. immitis p4, D. immitis nucleic acidsequence p22U, nucleic acid sequences including D. immitis p22U, andnucleic acid sequence comprising fragments of D. immitis p22U. Even morepreferred recombinant molecules include pβgal-p4, pHis-p4, pET19b-p4₆₃₅,pβgal-p22U, pHis-p22U, and pHis-p22U₆₀₈.

Recombinant cells of the present invention include any cells transformedwith any nucleic acid sequences of the present invention. Preferredrecombinant cells are transformed with recombinant molecules containingat least one of the following: a parasitic helminth nucleic acidsequence capable of hybridizing, under stringent conditions, to at leasta portion of Dirofilaria immitis nucleic acid sequence p4 or a parasitichelminth nucleic acid sequence capable of hybridizing, under stringentconditions, to at least a portion of Dirofilaria immitis nucleic acidsequence p22U. More preferred recombinant cells are transformed withrecombinant molecules including D. immitis nucleic acid sequence p4,nucleic acid sequences including D. immitis p4, nucleic acid sequencecomprising fragments of D. immitis p4, D. immitis nucleic acid sequencep22U, nucleic acid sequences including D. immitis p22U, and/or nucleicacid sequence comprising fragments of D. immitis p22U. Such recombinantcells can also be co-transformed with recombinant molecules includingnucleic acid sequences encoding other helminth parasitic proteins, suchas D. immitis P39, D. immitis P22L, D. immitis P20.5, D. immitis Di22,and D. immitis proteases expressed in L3 and/or L4 larvae, as well asother helminth proteins sharing significant homology with D. immitisP39, D. immitis P22L, D. immitis P20.5, D. immitis Di22 and D. immitisproteases expressed in L3 and/or L4 larvae. Di22 is disclosed in a FileWrapper Continuation, filed May 10, 1993, of U.S. patent applicationSer. No. 07/683,202, filed Apr. 8, 1991, entitled “Heartworm Vaccine”,which is incorporated by reference herein in its entirety. The proteaseis disclosed in U.S. patent application Ser. No. 07/792,209 filed Nov.12, 1991, entitled “Protease Vaccine Against Heartworm”, which isincorporated by reference herein in its entirety. Particularly preferredrecombinant cells include E. coli:pβgal-p4, E. coli:pHis-p4, E.coli:pET19b-p4₆₃₅ , E. coli:pβgal-p22U, E. coli:pHis-p22U and E.coli:pHis-p22U₆₀₈.

It may be appreciated by one skilled in the art that use of recombinantDNA technologies can improve expression of transformed nucleic acidsequences by manipulating, for example, the number of copies of thenucleic acid sequences within a host cell, the efficiency with whichthose nucleic acid sequences are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid sequences of the presentinvention include, but are not limited to, operatively linking nucleicacid sequences to high-copy number plasmids, integration of the nucleicacid sequences into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid sequences of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant enzyme productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing nucleic acid sequences encoding such a protein.

In accordance with the present invention, recombinant cells can be usedto produce at least one parasitic helminth protein of the present byculturing such cells under conditions effective to produce such aprotein, and recovering the protein. Effective conditions to produce aprotein include, but are not limited to, appropriate media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Anappropriate medium refers to any medium in which a cell of the presentinvention, when cultured, is capable of producing parasitic helminthproteins. An effective medium is typically an aqueous medium comprisingassimilable carbohydrate, nitrogen and phosphate sources, as well asappropriate salts, minerals, metals and other nutrients, such asvitamins. The medium may comprise complex nutrients or may be a definedminimal medium. Cells of the present invention can be cultured inconventional fermentation bioreactors, which include, but are notlimited to, batch, fed-batch, cell recycle, and continuous fermentors.Culturing can also be conducted in shake flasks, test tubes, microtiterdishes, and petri plates. Culturing is carried out at a temperature, pHand oxygen content appropriate for the recombinant cell. Such culturingconditions are well within the expertise of one of ordinary skill in theart. Examples of suitable conditions are included in the Examplessection.

Depending on the vector and host system used for production, resultantproteins may either remain within the recombinant cell; be secreted intothe fermentation medium; be secreted into a space between two cellularmembranes, such as the periplasmic space in E. coli; or be retained onthe outer surface of a cell or viral membrane. The phrase “recoveringthe protein” refers simply to collecting the whole fermentation mediumcontaining the protein and need not imply additional steps of separationor purification. Parasitic helminth proteins of the present inventioncan be purified using a variety of standard protein purificationtechniques, such as, but not limited to, affinity chromatography, ionexchange chromatography, filtration, electrophoresis, hydrophobicinteraction chromatography, gel filtration chromatography, reverse phasechromatography, chromatofocusing and differential solubilization.Isolated parasitic helminth proteins are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A vaccine for animals, forexample, should exhibit no substantial toxicity and should be capable ofstimulating the production of antibodies in a vaccinated animal.

One embodiment of the present invention is the expression of a parasitichelminth protein as a fusion protein which includes the parasitichelminth protein attached to a fusion segment. Such a fusion segmentoften aids in protein purification, such as permitting one to purify theresultant fusion protein using affinity chromatography. Fusion proteinscan be produced by culturing a recombinant cell transformed with afusion nucleic acid sequence that encodes a protein including the fusionsegment attached to either the carboxyl and/or amino terminal end of theparasitic helminth protein. Preferred fusion segments include, but arenot limited to, glutathione-S-transferase, β-galactosidase, apolyhistidine segment capable of binding to a divalent metal ion,maltose binding protein and immunoglobulin binding domains (e.g.,protein A or portions thereof) with a polyhistidine segment being morepreferred. Examples of fusion proteins of the present invention includePβGAL-P4, PHIS-P4, PHIS-P4₆₃₅, PβGAL-P22U, PHIS-P22U and PHIS-P22U₆₀₈.

The present invention also includes antibodies capable of selectivelybinding to a parasitic helminth protein or mimetope thereof, the proteinor mimetope thereof being capable of selectively binding to at least onecomponent of anti-parasitic helminth immune serum. Such antibodies canbe either polyclonal or monoclonal antibodies. Antibodies of the presentinvention include functional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies,that are capable of selectively binding to at least one of the epitopesof the protein or mimetope used to obtain the antibodies. Preferredantibodies are raised in response to proteins, or mimetopes thereof,that are encoded, at least in part, by a nucleic acid sequence capableof hybridizing, under stringent conditions, to at least a portion ofDirofilaria immitis nucleic acid sequence p4 or to at least a portion ofDirofilaria immitis nucleic acid sequence p22U.

A preferred method to produce antibodies of the present inventionincludes administering to an animal an effective amount of an isolatedprotein or mimetope thereof to produce the antibody, wherein the proteinis capable of selectively binding to at least one component of serumfrom an animal that is immune to infection by the helminth, the serumbeing capable of inhibiting helminth development; and recovering theantibodies. Preferably the protein is encoded, at least in part, by aparasitic helminth nucleic acid sequence capable of hybridizing, understringent conditions, to at least a portion of Dirofilaria immitisnucleic acid sequence p4 or by a parasitic helminth nucleic acidsequence capable of hybridizing, under stringent conditions, to at leasta portion of Dirofilaria immitis nucleic acid sequence p22U. Antibodiesraised against defined proteins or mimetopes can be advantageous becausesuch antibodies are not substantially contaminated with antibodiesagainst other substances that might otherwise cause interference in adiagnostic assay or side effects if used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as vaccines to passively immunize an animalin order to protect the animal from parasitic helminth infections, (b)as reagents in assays to detect parasitic helminth infection, and/or (c)as tools to recover desired parasitic helminth proteins from a mixtureof proteins and other contaminants.

Furthermore, antibodies of the present invention can be used to targetcytotoxic agents to parasitic helminths in order to directly killhelminths expressing proteins selectively bound by the antibodies.Targeting can be accomplished by conjugating (i.e., stably joining) suchantibodies to the cytotoxic agents. Suitable cytotoxic agents include,but are not limited to: double-chain toxins (i.e., toxins having A and Bchains), such as diphtheria toxin, ricin toxin, Pseudomonas exotoxin,modeccin toxin, abrin toxin, and shiga toxin; single-chain toxins, suchas pokeweed antiviral protein, α-amanitin, and ribosome inhibitingproteins; and chemical toxins, such as melphalan, methotrexate, nitrogenmustard, doxorubicin and daunomycin. Preferred double-chain toxins aremodified to include the toxic domain and translocation domain of thetoxin but lack the toxin's intrinsic cell binding domain.

One embodiment of the present invention is a therapeutic compositioncapable of protecting an animal from parasitic helminth infection whenadministered to the animal in an effective manner. Such a compositionincludes at least one of the following protective compounds: (a) anisolated parasitic helminth protein, or mimetope thereof, capable ofselectively binding to at least one component of an anti-parasitichelminth immune serum such that the protein is preferably encoded, atleast in part, by a nucleic acid sequence capable of hybridizing, understringent conditions, to at least a portion of D. immitis nucleic acidsequence p4; (b) an isolated parasitic helminth protein, or mimetopethereof, capable of selectively binding to at least one component of ananti-parasitic helminth immune serum such that the protein is preferablyencoded, at least in part, by a nucleic acid sequence capable ofhybridizing, under stringent conditions, to at least a portion of D.immitis nucleic acid sequence p22U; (c) an antibody capable ofselectively binding to a parasitic helminth protein or mimetope thereof,that is capable of selectively binding to at least one component of ananti-parasitic helminth immune serum such that the protein is preferablyencoded, at least in part, by a nucleic acid sequence capable ofhybridizing, under stringent conditions, to at least a portion of D.immitis nucleic acid sequence p4; (d) an antibody capable of selectivelybinding to a parasitic helminth protein or mimetope thereof, that iscapable of selectively binding to at least one component of ananti-parasitic helminth immune serum such that the protein is preferablyencoded, at least in part, by a nucleic acid sequence capable ofhybridizing, under stringent conditions, to at least a portion of D.immitis nucleic acid sequence p22U; (e) an isolated parasitic helminthnucleic acid sequence capable of hybridizing, under stringentconditions, to at least a portion of D. immitis nucleic acid sequencep4; and/or (f) an isolated parasitic helminth nucleic acid sequencecapable of hybridizing, under stringent conditions, to at least aportion of D. immitis nucleic acid sequence p22U.

Administration of a therapeutic composition containing multipleprotective compounds targeting multiple parasitic helminths to an animalcan protect the animal from infection by those helminths. Similarly,administration of a therapeutic composition targeting different aspectsof a given parasitic helminth may provide additional protection to theanimal. For example, a therapeutic composition of the present inventioncan include at least one of the following additional compounds: D.immitis P39, D. immitis P22L, D. immitis P20.5, D. immitis Di22, and D.immitis proteases expressed in L3 and/or L4 larvae, as well as otherhelminth proteins sharing significant homology with D. immitis P39, D.immitis P22L, D. immitis P20.5, D. immitis Di22, and D. immitisproteases expressed in L3 and/or L4 larvae, as well as mimetopes of suchproteins, antibodies that selectively bind to such proteins or mimetopesthereof, and nucleic acid sequences encoding such proteins.

As used herein, a protective compound refers to a compound that whenadministered to an animal in an effective manner is able to treat,ameliorate, and/or prevent infection by a parasitic helminth. Preferredhelminths are heretofore disclosed.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals, and more preferably to dogs, cats,humans, ferrets, horses, cattle, sheep and other pets and/or economicfood animals. Preferred animals to protect include dogs, cats, humansand ferrets, with dogs and cats being particularly preferred.

Therapeutic compositions of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, m or o-cresol, formalin and benzylalcohol. Standard formulations will either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientmay comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline could be added prior to administration.

In one embodiment of the present invention, the therapeutic compositioncan also include an immunopotentiator, such as an adjuvant or a carrier.Adjuvants are typically substances that generally enhance the immuneresponse of an animal to a specific antigen. Suitable adjuvants include,but are not limited to, Freund's adjuvant; other bacterial cell wallcomponents; aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins; viral coat proteins; otherbacterial-derived preparations; gamma interferon; block copolymeradjuvants, such as Hunter's Titermax adjuvant (Vaxcel™, Inc. Norcross,Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives, such as Quil A(available from Superfos Biosector A/S, Denmark). Carriers are typicallycompounds that increase the half-life of a therapeutic composition inthe treated animal. Suitable carriers include, but are not limited to,polymeric controlled release formulations, biodegradable implants,liposomes, bacteria, other viruses, oils, esters, and glycols.

In order to protect an animal from parasitic helminth infection, atherapeutic composition of the present invention is administered to theanimal in an effective manner such that the composition is capable ofprotecting that animal from infection. For example, an isolated proteinor mimetope thereof, when administered to an animal in an effectivemanner, is able to elicit (i.e., stimulate) an immune response,preferably including both a humoral and cellular response, that issufficient to protect the animal from infection. Similarly, an antibodyof the present invention, when administered to an animal in an effectivemanner, is administered in an amount so as to be present in the animalat a titer that is sufficient to protect the animal from infection, atleast temporarily. Nucleic acid sequences of the present invention,preferably oligonucleotides, can also be administered in an effectivemanner, thereby reducing expression of parasitic helminth proteins inorder to interfere with parasite development.

Therapeutic compositions of the present invention can be administered toanimals prior to parasite infection in order to prevent infection and/orcan be administered to animals after parasite infection in order totreat disease caused by the parasite. For example, proteins, mimetopesthereof, and antibodies thereof can be used as immunotherapeutic agents.

Acceptable protocols to administer therapeutic compositions in aneffective manner include individual dose size, number of doses,frequency of dose administration, and mode of administration.Determination of such protocols can be accomplished by those skilled inthe art. A suitable single dose is a dose that is capable of protectingan animal from parasitic helminth infection when administered one ormore times over a suitable time period. For example, a preferred singledose of a protein, mimetope or antibody therapeutic composition is fromabout 1 microgram (μg) to about 10 milligrams (mg) of the therapeuticcomposition for an animal about the size of a dog. Booster vaccinationscan be administered from about 2 weeks to several years after theoriginal administration. Preferably booster vaccinations areadministered when the immune response of the animal becomes insufficientto protect the animal from parasitic helminth infection. A preferredadministration schedule is one in which from about 10 μg to about 1 mgof the vaccine per kg body weight of the animal is administered fromabout one to about two times over a time period of from about 2 weeks toabout 12 months. Modes of administration can include, but are notlimited to, subcutaneous, intradermal, intravenous, nasal, oral,transdermal and intramuscular routes.

According to one embodiment, nucleic acid sequences of the presentinvention can also be administered to an animal in a fashion to enableexpression of the nucleic acid sequence into a protective protein in theanimal to be protected from parasitic helminth infection. Nucleic acidsequences can be delivered in a variety of methods including, but notlimited to, direct injection (e.g., as “naked” DNA or RNA molecules,such as is taught, for example in Wolff et al., 1990, Science 247,1465-1468), packaged as a recombinant virus particle vaccine, andpackaged as a recombinant cell vaccine.

A recombinant virus particle vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging-deficient. A number ofrecombinant virus particles can be used, including, but not limited to,those based on alphaviruses, pox viruses, adenoviruses, herpes viruses,and retroviruses. Preferred recombinant particle viruses are those basedon alphaviruses, with those based on Sindbis virus, Semliki virus, andRoss River virus being more preferred. Methods to produce and userecombinant virus particle vaccines are disclosed in U.S. patentapplication Ser. No. 08/015,414, filed Feb. 8, 1993, entitled“Recombinant Virus Particle Vaccines”, which is incorporated byreference herein in its entirety.

When administered to an animal, the recombinant virus particle vaccineinfects cells within the immunized animal and directs the production ofa parasitic helminth protein or RNA that is capable of protecting theanimal from infection by the helminth. For example, when the helminthprotein is a D. immitis protein, the recombinant virus particle vaccineis administered according to a protocol that results in the animalproducing a sufficient immune response to protect itself from heartworm.A preferred single dose of a recombinant virus/particle vaccine of thepresent invention is from about 1×10⁴ to about 1×10⁵ virus plaqueforming units (pfu) per kilogram body weight of the animal.Administration protocols are similar to those described herein forprotein-based vaccines.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express at least one parasitichelminth protein. Preferred recombinant cells include Salmonella,Escherichia coli, and Mycobacterium recombinant cells, with Salmonellarecombinant cells being more preferred. Such recombinant cells can beadministered in a variety of ways but have the advantage that they canbe administered orally, preferably at doses ranging from about 10⁸ toabout 10¹² bacteria per kilogram body weight. Administration protocolsare similar to those described herein for protein-based vaccines. Incommon with most other enteric pathogens, Salmonella strains normallyenter the host orally. Once in, the intestine, they interact with themucosal surface, normally to establish an invasive infection. MostSalmonella infections are controlled at the epithelial surface, causingthe typical Salmonella-induced gastroenteritis. Some strains ofSalmonella, including S. typhi and some S. typhimurium isolates, haveevolved the ability to penetrate deeper into the host, causing adisseminated systemic infection. It appears such strains have thecapacity to resist the killing actions of macrophages and other immunecells. S. typhi can exist for long periods as a facultativeintracellular parasite. Some of the live vaccine strains can alsopersist for long periods in the mononuclear phagocyte system. Hostsinfected in such a manner develop, in addition to a mucosal immuneresponse, systemic cellular and serum antibody responses to theSalmonella. Thus, invading Salmonella, whether virulent or attenuated,can stimulate strong immune responses, unlike many other entericpathogens which only set up local, noninvasive gut infections. Thepotent immunogenicity of live Salmonella makes them attractivecandidates for carrying parasitic helminth proteins to the immunesystem.

A preferred recombinant cell-based vaccine is one in which the cell isattenuated. Salmonella typhimurium strains, for example, can beattenuated by introducing mutations into genes critical for in vivogrowth and survival. For example, genes encoding cyclic adenosinemonophosphate (cAMP) receptor protein or adenylate cyclase are deletedto produce avirulent, vaccine strains. Such strains can deliver antigensto lymphoid tissue in the gut but demonstrate reduced capacity to invadethe spleen and mesenteric lymph nodes. These strains will stillstimulate both humoral and cellular immunity in mammalian hosts.

Recombinant cell vaccines can be used to introduce isolated proteins ofthe present invention into the immune systems of animals. For example,recombinant molecules comprising parasitic helminth nucleic acidsequences of the present invention operatively linked to expressionvectors that function in Salmonella can be transformed into Salmonellahost cells. The resultant recombinant cells are then introduced into theanimal to be protected. Preferred Salmonella host cells are those forwhich survival depends on their ability to maintain the recombinantmolecule (i.e., a balanced-lethal host-vector system). An example ofsuch a preferred host/recombinant molecule combination is a Salmonellastrain (e.g., UK-1 _(x)3987 or SR-11 _(x)4072) which is unable toproduce aspartate β-semialdehyde dehydrogenase in combination with arecombinant molecule also capable of encoding the enzyme. Aspartateβ-semialdehyde dehydrogenase, encoded by the asd gene, is an importantenzyme in the pathway to produce diaminopimelic acid (DAP). DAP is anessential component of the peptidoglycan of the cell wall ofGram-negative bacteria, such as Salmonella, and, as such, is necessaryfor survival of the cell. Thus, Salmonella lacking a functional asd genecan only survive if they maintain a recombinant molecule that is alsocapable of expressing a functional asd gene.

The efficacy of a therapeutic composition of the present invention toprotect an animal from infection by a parasitic helminth can be testedin a variety of ways including, but not limited to, detection ofprotective antibodies (using, for example, proteins or mimetopes of thepresent invention), detection of cellular immunity within the treatedanimal, or challenge of the treated animal with the parasitic helminthor antigens thereof to determine whether the treated animal is resistantto infection. Such techniques are known to those skilled in the art.

One preferred embodiment of the present invention is the use of D.immitis nucleic acids and proteins to protect an animal from heartworminfection. It is particularly preferred to prevent L3 larvae that aredelivered to the animal by the mosquito intermediate host from maturinginto adult worms. As such, preferred therapeutic compositions are thosethat are able to inhibit at least one step in the portion of theparasite's development cycle that includes L3 larvae, third molt, L4larvae, fourth molt, immature adult prior to entering the circulatorysystem. In dogs, this portion of the development cycle is about 70 days.As such, preferred nucleic acid sequences, proteins, and antibodies toprotect an animal against heartworm include D. immitis p4 and D. immitisp22U, as well as nucleic acid sequences including at least a portion ofD. immitis p4 and/or D. immitis p22U, proteins encoded by thosesequences, mimetopes of such proteins, and antibodies that selectivelybind to such proteins. Particularly preferred therapeutic compositionsinclude proteins that share at least some D. immitis P4 and/or D.immitis P22U epitopes. Such compositions are administered to animals ina manner effective to protect the animals from heartworm infection.Additional protection may be obtained by administering additionalprotective compounds, including other D. immitis antigens, such as D.immitis P39, D. immitis P22L, D. immitis P20.5, D. immitis Di22, and/orD. immitis proteases expressed in L3 and/or L4 larvae.

It is also within the scope of the present invention to use the isolatedparasitic helminth proteins, mimetopes, nucleic acid sequences, andantibodies as diagnostic agents. Preferably such diagnostic agents aresupplemented with additional compounds that can detect other phases ofthe helminth's life cycle.

One embodiment of the present invention is a therapeutic compositioncapable of protecting an animal from parasitic helminth infection whenadministered to the animal in an effective manner that includes acompound capable of substantially interfering with the function of aparasitic helminth protein LDLr class A cysteine-rich motif, preferablyby reducing the ability of such a protein to take up sterols. As usedherein, a parasitic helminth protein LDLr class A cysteine-rich motif,or LDLr class A motif, refers to cysteine-rich motifs in parasitichelminth proteins that are homologous to that identified in D. immitisP4. Such motifs also occur in several other proteins, including LDLreceptor-related proteins and α₂-macroglobulin receptors, as heretoforedisclosed. As used herein, substantially interferes refers to theability of the compound to inhibit parasitic helminth development.

Preferred therapeutic compositions are those that are targeted to theLDLr class A motif shared by D. immitis P4 and other parasitic helminthproteins encoded, at least in part, by a nucleic acid sequence capableof hybridizing, under stringent conditions, to at least a portion of D.immitis p4. Suitable compounds can be identified by a variety ofmethods, including known methods to screen inorganic and organicmolecules and rational drug design methods in which the active site ofthe motif is identified and a compound designed that would interferewith that active site. Suitable compounds are likely to include sterolmimetopes that are capable of interfering with sterol uptake byparasitic helminths, possibly by selectively binding to the LDLr class Amotif.

Parasitic helminths, some protozoans and some insects are not able tosynthesize squalenes and sterols de novo. Thus, parasitic helminthsrequire sterols as precursors for steroid hormones and as integralstructural components of cellular membranes. Cholesterol, one of thesterols that parasitic helminths cannot produce de novo, regulatescellular function, growth and differentiation by interacting with anumber of protein kinases, protein receptors and ion pumps. Cholesterolis also the precursor of ecdysteroids, the steroidal molting hormones ofinsects, also believed to serve a similar function in parasitichelminths. While not being bound by theory, it is believed that the LDLrclass A motif is important in the development of parasitic helminths(including nematodes, trematodes, and cestodes) as well as otherorganisms that do not synthesize sterols de novo (e.g., some parasiticprotozoans and insects), because known LDLr class A motifs areapparently involved in sterol uptake. Such motifs in LDL receptors, forexample, are responsible for binding the positively-charged ligandsapolipgprotein B (apo B) and apolipoprotein E (apo E) within lipoproteinparticles (see, for example, Herz et al., 1988, EMBO J. 7, p.4119-4127). Apo E is involved in the clearance of triglyceride-richlipoproteins and in reverse cholesterol transport. ApoE is also thoughtto be involved in the modulation of cell growth in mammalian lymphocytesas well as in brain and other tissues. Thus, compounds having theability to interfere with sterol uptake by parasitic helminths due totheir ability to interact with LDLr class A motifs are attractive astherapeutic compositions of the present invention.

Such therapeutic compositions can be administered to animals in aneffective manner to protect animals from parasitic helminth infection.Effective amounts and dosing regimens can be determined using techniquesknown to those skilled in the art.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the invention.

EXAMPLES Example 1

This Example describes a procedure for producing and evaluating immunesera of the present invention.

Four dogs were immunized with chemically-abbreviated D. immitis larvalinfections (using the method described in Grieve et al., 1988, ibid.),and two dogs served as chemically-treated controls. The dogs were housedin indoor mosquito-free individual cages at a temperature of about 22°C. and about 40% to about 65% humidity. On day 532, post initialimmunization, each dog was challenged with about 100 L3 D. immitislarvae by implanting 5 diffusion chambers per dog, each diffusionchamber containing about 20 L3 D. immitis larvae, using the methoddescribed in Grieve et al., 1988, ibid. Concomitant with chamberimplantation, each dog was injected subcutaneously with about 50 L3 D.immitis larvae, and the infection was allowed to proceed beyond theanticipated prepatent period. Challenge infections were repeated on day588, post initial immunization, both by implanting 5 diffusion chambersper dog, each chamber having about 20 L3 D. immitis larvae and bysubcutaneously inoculating about 30 L3 D. immitis larvae per dog. Serumsamples were collected from the immunized dogs at numerous time pointsthroughout the study period. Serum samples were analyzed for antibodiesthat selectively bound to L3 and/or L4 surface antigens using anindirect fluorescent antibody assay, and for antibodies that selectivelybound to L3 soluble antigens, L4 soluble antigens and/or to anexcretory/secretory antigen fraction using an indirect ELISA, asdescribed by Grieve et al., 1988, ibid. The results indicated that serumfrom dogs that had been immunized and challenged with D. immitis larvaehad produced antibodies to both surface and soluble D. immitis larvalantigens. The sera were pooled, and those obtained from larval-immunizeddogs (i.e., anti-larval immune sera) were shown to inhibit larvaldevelopment; see, for example, Example 2. Immune sera were also shown toselectively bind to L3 and/or L4 larval proteins having molecularweights of about 15 kD, 23/24 kD doublet, 31 kD, 33 kD, 39 kD, 42 kD, 55kD, 59 kD, 66 kD, 70 kD, 97 kD and 207 kD by Tris-glycine SDS PAGE.

Example 2

This Example demonstrates that serum collected from larval-immunizeddogs, produced as described in Example 1, is capable of inhibitingparasite development whereas serum collected from non-immunized dogs isnot.

One subcutaneous pocket was formed in each of about 3 to about 6 Balb/CBYJ mice that were about 10 weeks old. One diffusion chamber, containing20 L3 D. immitis larvae, was implanted into each pocket alone with 0.5ml of sera collected from immunized dogs or from non-immunized dogs,produced as described in Example 1. The diffusion chambers wererecovered two or three weeks later. Living larvae in the chambers werecounted and placed into glacial acetic acid, followed by 70% ethanolcontaining 5% glycerin. The ethanol was allowed to evaporate leaving thelarvae in glycerin. The larvae were measured using projected images inthe Macmeasure image analysis system on a Macintosh computer.

Three experiments, in which different serum samples were exposed tolarvae in diffusion chambers, were conducted: Experiment 1 comparedequal portions of sera collected from individual dogs at days 56, 77 and117 after challenge. Experiments 2 and 3 compared serum collected fromimmunized dogs 117 days after initial challenge to control sera. Inexperiment 2, the control serum was a pool of sera collected from 12naive dogs; in experiment 3, control serum was collected from a singlenaive dog. Each of the experiments also included controls in which thelarvae were not exposed to any serum.

In experiment 1, chambers were recovered two weeks post-inoculation. Thenumber of larvae retrieved from chambers implanted in mice receivingserum from immunized (i.e., immune) dogs was lower than that of larvaein chambers implanted in mice receiving naive dog serum, but thedifference was not statistically significant. Also, no differences wereseen between the length of larvae regardless of which serum was used.

In experiments 2 and 3, the chambers were recovered three weeks afterinfection. There were significant differences in the larval recoveriesbetween those receiving serum from naive dogs and those from immunedogs; there were about 34% more larvae recovered from mice treated withnaive dog serum than were recovered from mice treated with immune serum.The lengths of the larvae were also significantly shorter in thosechambers exposed to sera from immune dogs compared to larvae in chambersexposed to naive dog sera. Thus, this Example shows that serum collectedfrom dogs immune to D. immitis infection inhibits larval development,compared to serum collected from naive dogs.

Example 3

This Example describes the purification of D. immitis P22U as well astryptic digestion of the protein, and partial amino acid sequencing ofseveral tryptic fragments.

Third stage larvae were collected and cultured in vitro as described inFrank et al., 1992, ibid. The larvae were washed free of serum proteinsat about 48 hr, placed back into culture and the serum-free mediacontaining larval ES products was collected from 48 to 144 hr inculture. Each week's yield of ES was collected, filtered through a 0.45μm filter (Acrodisc™, Gelman Sciences, Ann Arbor, Mich.) and frozen atabout −70° C. until further processing. Processing was conducted atabout 4° C. or on ice and consisted of thawing the ES and adding 0.5 MEDTA.Na₂, pH 8.0, to a final concentration of 5 mM. EDTA was the onlyprotease inhibitor used since only metalloprotease activity has beenfound in larval ES (Richer et al., 1992, Exp. Parasit. 75, p. 213-222).The ES was concentrated and the buffer was exchanged using Centriprep-10and Centricon-10 (Amicon, Beverly, Mass.); the final buffer was 20 mMTris, 1 mM EDTA.Na., pH 7.2.

All chromatography was performed on a Beckman 338 binary system usingSystem Gold version 3.10 chromatography software (Beckman Instruments,Inc., San Ramon, Calif.). The separations and fraction collections wereconducted at room temperature and the fractions placed at about 4° C.immediately after each run. When portions of the samples weremetabolically labeled, aliquots of the collected fractions were assayedin scintillation fluid by a Beckman Model LS 1801 liquid scintillationcounter (Beckman Instruments, Inc.).

The first purification was from approximately 38,650 larvae, 3,550 ofwhich had been metabolically labeled with Translabel™ from about 48 to144 hr. The ES products were concentrated to 175 μl in 20 mM Tris, 1 mMEDTA.Na₂, pH 7.2 (Buffer A) and contained 1.3 μg/μl protein with an³⁵S-incorporation of 7,450 cpm/μl. Cation exchange chromatography wasused as the first step in purification. A SynChropak CM300-GRD 4.6×50 mmcolumn (Synchrom, Inc., Lafayette, Ind.) was used. The sample wasdiluted with 300 μl buffer A, centrifuged at 12,000 g and the supernateinjected onto the column at 0.5 ml/min Buffer A. After a 5 min wash, theadsorbed proteins were eluted with a steep gradient to 100% Buffer B (1M KCl in Buffer A) over 0.1 min while 200 μl fractions were collectedthroughout. Detection of proteins was at 280 nm. FIG. 1 shows theresultant chromatogram. Boxed fractions, designated 4, 5, 6, 23, 24, 25and 26, were evaluated by SDS PAGE.

The vast majority of contaminating proteins eluted in the initial peak.In contrast, P22U, as well as P22L and P20.5, eluted in the second peak,i.e., in fractions 23, 24, 25 and 26.

Reverse phase chromatography using a Vydac C₄ 0.21×25 cm, 5 μm particlesize column (Vydac 214TP52, The Separations Group, Hesperia, Calif.) wasused to separate P22U from P22L and P20.5. Buffer C consisted of 0.1%trifluoroacetic acid (TFA), 0.085% triethylamine (TEA) in Milli-Q waterproduced by processing 18 megaohm water through a Milli-Q Plus watersystem (Millipore Corp., Bedford Mass.), while Buffer D consisted of0.085% TFA, 0.085% TEA, 80% CH₃CN in Milli-Q water. Detection ofproteins was at 220 nm. Fractions 23 and 24 from the cation exchange runwere injected onto the column followed by fractions 25 and 26 two minlater. The initial flow rate was 0.25 ml/min at 12.5% D, 87.5% C. Theflow rate was reduced to 0.17 ml/min at 4 min and a gradient to 62.5% Dover 200 min was started at 6 min. Fractions of 0.75 min were collected.

Aliquots of peak fractions were submitted to SDS-PAGE and analyzed bysilver staining and autoradiography. P20.5 appeared first andpredominated in fractions 99-102 (elution times of from about 74.25minutes through about 76.5 minutes). P22L predominated in fractions103-107, (elution times of from about 77.25 minutes through about 80.25minutes), although there was significant contamination with P20.5. P22Ueluted much later, in fractions 229-235 (elution times of from about171.75 minutes through about 176.25 minutes).

Purified P22U obtained from C₄ reverse phase chromatography wasdenatured, reduced and pyridylethylated by standard procedures (see, forexample, Matsudaira, P. T. (ed.)., 1989, A Practical Guide to Proteinand Peptide Purification for Microsequencing). The pyridylethylated P22Uwas subjected to trypsin digestion, and the tryptic peptides separatedby C₁₈ reverse phase chromatography using a 0.21-cm×25-cm, 5-μm particlesize column (Vydac 218TP52) by a procedure based on Stone et al., 1989,in Matsudaira, P. T. (ed.)., A Practical Guide to Protein andPurification for Microsequencing, p. 31-47.

The chromatogram depicting the tryptic fragments of P22U is shown inFIG. 2. The fragments indicated by asterisks were submitted forsequencing. All sequencing was conducted at Macromolecular Resources,Department of Biochemistry, Colorado State University, Fort Collins,Colo. The peptides were concentrated to 50 μl or less using a Speedvac®and frozen at about −20° C. until sequencing. N-terminal sequencing wasconducted in an ABI Model 473A Protein/Peptide Sequencer System (AppliedBiosystems, Inc., Foster City, Calif.) using pulsed liquid chemistry andon line microgradient PTH amino acid analysis (see, for example, Hewicket. al., 1981, J. Biol. Chem. 256, p. 7990-7997; Geisow and Aitken,1989, in Findlay, J. B. C. and M. J. Geisow (ed.). Protein Sequencing: APractical Approach, p. 85-98). The most likely sequence of the trypticfragment eluting at 44 minutes (referred to as the 44 min trypticfragment), using one-letter amino acid code, was MAQDAFPNACAQGEPK (SEQID NO:6). The most likely sequence of the tryptic fragment eluting at 58minutes (referred to as the 58 min tryptic fragment) wasAIAPCQLTAVQSVLPCADQCQK (SEQ ID NO:7). The most likely sequence of thetryptic fragment eluting at 60 minutes (referred to as the 60 mintryptic fragment) was LGSCSPDCGLDLPSDNVMVQDV (SEQ ID NO:8).

Example 4

This Example describes the cloning and sequencing of D. immitis nucleicacid sequence p4. D. immitis p4 was identified by its ability to encodea protein that selectively bound to at least one component of immuneserum collected from a dog immunized with D. immitis larvae.

D. immitis L3 larvae were harvested from mosquitos using standardtechniques and cultivated in vitro in 50:50 NCTC-135/IMDM (NI) media(Sigma) supplemented with 20% serum supplement at 37° C., 5% carbondioxide for 48 hours. Total RNA was extracted from the larvae using anacid-guanidinium-phenol-chloroform method similar to that described byChomczynski and Sacchi, 1987, Anal. Biochem. 162, p. 156-159.Approximately 15,000 to 30,000 larvae were used in an RNA preparation.Poly A+ selected RNA was separated from total RNA by oligo-dT cellulosechromatography using Oligo dT cellulose from Collaborative Research,Inc., Waltham, Mass., according to the method recommended by themanufacturer.

A D. immitis L3 larval cDNA expression library was constructed in lambda(λ) Uni-ZAP™ XR vector (available from Stratagene Cloning Systems, LaJolla, Calif.) using Stratagene's ZAP-cDNA Synthesis Kit® protocol andabout 5 μg to about 6 μg of L3 poly A+. The resultant library wasamplified to a titer of about 4.88×10⁹ pfu/ml with about 97%recombinants.

Using the protocol described in the Stratagene picoBlue immunoscreeningkit, the L3 larval cDNA expression library was screened with immune dogserum prepared as described in Example 1. Antibodies specific for ahighly immunoreactive protein termed the “ladder protein” (Culpepper etal., 1992, Mol. Biochem. Parasitol. 54, p. 51-62) had been adsorbed fromthis serum by affinity chromatography with a recombinant GST-ladderfusion protein. Immunoscreening of duplicate plaque lifts of the cDNAlibrary with the same serum identified 4 positive clones, one of whichincluded D. immitis nucleic acid sequence p4. The remaining 3 cloneswere shown to encode at least portions of P39, as disclosed in U.S.patent application Ser. No. 08/003,389, referenced above.

The plaque-purified clone including D. immitis nucleic acid sequence p4was converted into a double stranded recombinant molecule, hereindenoted as pβgal-p4, using R408 helper phage and XL1-Blue E. coliaccording to the in vivo excision protocol described in the StratageneZAP-cDNA Synthesis Kit. Double stranded plasmid DNA was prepared usingan alkaline lysis protocol, such as that described in Sambrook et al.,ibid. The plasmid DNA was digested with EcoRI and XhoI restrictionendonucleases to release two D. immitis DNA fragments of about 580 and320 nucleotides, the entire D. immitis p4 fragment being about 900nucleotides in size.

The plasmid containing D. immitis p4, was sequenced using the Sangerdideoxy chain termination method, as described in Sambrook et al., ibid.The Promega Erase a Base method (available from Promega Corp., Madison,Wis.) was used to generate deletion clones for sequence analysis. Anabout 913-nucleotide consensus sequence of the entire D. immitis p4 DNAfragment was determined and is presented as SEQ ID NO:1. The entire 913nucleotides form an open reading frame encoding an amino acid sequenceof about 303 amino acids, presented in SEQ ID NO:2. The first ATG codonwithin this sequence spans nucleotides from about 417 through about 419.As such, SEQ ID NO:1 does not encode a full-length protein, but doesencode a protein that selectively binds to at least one component ofimmune dog serum. The predicted size of the protein encoded by SEQ IDNO:1 is about 35.5 kD, with an estimated pI of about 4.26.

A homology search of the non-redundant protein sequence database wasperformed through the National Center for Biotechnology Informationusing the BLAST network. This database includesSwissProt+PIR+SPUpdate+GenPept+GPUpdate. The search was performed usingSEQ ID NO:2 and showed the only significant homology shared between SEQID NO:2 and known sequences to be a contiguous stretch of 9 amino acids,namely DDCGDGSDE (SEQ ID NO:5), that was also found in humanLDL-receptor related protein, human and mouse alpha-2-macroglobulinreceptors and rat renal GP 330 glycoprotein. A conserved stretch ofeight of the nine amino acids is also found in Caenorhabditis elegansLDL receptor-related protein and C. elegans basement membraneproteoglycan.

Example 5

This example demonstrates the ability of D. immitis p4 to encode aprotein that selectively binds to immune serum.

Recombinant molecule pET19b-p4₆₃₅, containing D. immitis p4 nucleotidesfrom about 1 through about 635 operatively linked to bacteriophage T7lactranscription control sequences and to a fusion sequence encoding apoly-histidine segment comprising 10 histidines was produced in thefollowing manner. An about 635-nucleotide DNA fragment containingnucleotides spanning from about 1 through about 635 of SEQ ID NO:1,called p4₆₃₅, was PCR amplified from a clone containing D. immitis p4using the primers 5′ CGGGATCCCGAGTTAAATAGTCG 3′ (denoted SEQ ID NO:9 or394-5′; BamHI site underlined) and 5′ TGCAGGATCCTGCACCG 3′ (denoted SEQID NO:10 or 394-3′; BamHI site underlined). The PCR product was digestedwith BamHI restriction endonuclease, gel purified and subcloned intoexpression vector pET19b (available from Novagen Inc., Madison, Wis.)that had been cleaved with BamHI. The resulting recombinant moleculepET19b-p4₆₃₅ was transformed into E. coli BL21(DE3)pLysS to formrecombinant cell E. coli:pET19b-p4₆₃₅ . E. coli BL21(DE3)pLysS includesa bacteriophage T7 RNA polymerase gene under the control of lactranscription control sequences.

Recombinant cell E. coli:pET19b-p4₆₃₅ was cultured in shake flaskscontaining an enriched bacterial growth medium containing 0.1 mg/mlampicillin at about 37° C. When the cells reached an optical density atabout 600 nanometers (OD₆₀₀) of about 0.819, expression of D. immitis p4was induced by addition of about 1 mM isopropyl-β-D-thiogalactoside(IPTG). Protein production was monitored by SDS PAGE of recombinant celllysates, followed by Coomassie blue staining, using standard techniques.Recombinant cell E. coli:pET19b-p4₆₃₅ produced a protein, denotedPHIS-P4₆₃₅, that migrated with an apparent molecular weight of about 37kD. Such a protein was not produced by cells transformed with thepET-19b plasmid lacking a D. immitis DNA insert.

Immunoblot analysis of recombinant cell E. coli:pET19b-p4₆₃₅ lysatesindicates that the 37 kD protein is able to selectively bind to immunedog serum and, as such, is capable of binding to at least one componentof a serum that is capable of inhibiting D. immitis larval development.

The E. coli:pET19b-p4₆₃₅ histidine fusion peptide was separated fromsoluble E. coli proteins by nickel chelation chromatography and animidazole gradient. Immunoblot analysis of the total E.coli:pET19b-p4₆₃₅ lysate, column eluate and column void volume indicatesthat the 37 kD protein can be isolated on the nickel column and is ableto selectively bind to immune dog serum, and as such, is capable ofbinding to at least one component of a serum that is capable ofinhibiting D. immitis larval development. The column eluate was notdetected by preimmune sera from the same immune dog.

Example 6

This Example describes the isolation and sequence of D. immitis nucleicacid sequence p22U.

Total RNA was extracted from adult female D. immitis worms, poly A+ RNAprepared, and an adult female D. immitis cDNA library produced, usingmethods similar to those described in Example 4.

A segment of DNA for use in the identification of a nucleic acidsequence capable of encoding at least a portion of P22U was produced byPCR amplification using standard techniques, such as those described inSambrook et al., ibid. Briefly, first strand cDNA was synthesized fromadult female poly A+ RNA using Murine Leukemia Virus reversetranscriptase (available from Stratagene) and Stratagene's linker-primerfrom their ZAP-cDNA Synthesis Kit, namely 5′GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTT-TTTTTTTTT 3′ (SEQ ID NO:11). Apool of two sets of degenerate primers was produced based on the partialamino acid sequence of the 60 min tryptic fragment described in Example3. One degenerate set of primers, denoted GRF 11, includes the followingsequences: 5′TGYTCNCCNGAYTGYGG 3′ (SEQ ID NO:12), wherein Y can beeither C or T, and N can be either A, G, C or T. The second set ofprimers, denoted GRF 12, includes the following sequences: 5TGYAGTCCNGAYTGYGG 3′ (SEQ ID NO:13). PCR amplification using the pool ofdegenerate primers in combination with Stratagene's linker-primer as theantisense primer was used to amplify the DNA segment. Verification thatthe appropriate segment had been amplified was accomplished by Southernblot analysis using a degenerate probe based on a more C-terminal aminoacid sequence of the 60 min tryptic fragment, namely GRF 3 whichincludes the following sequences: 5′ TGNACCATNACRTTRTC 3′ (SEQ IDNO:14), wherein R can be either A or G.

The amplified segment was gel purified, electroeluted and cloned intothe pCR II cloning vector (available from Invitrogen, San Diego,Calif.), following the manufacturers' instructions. Two clones werepartially sequenced, yielding a nucleic acid sequence which included asequence corresponding to the amino acid sequence of the 60 min trypticfragment. The nucleic acid sequence includes from nucleotides about 444to about 696 of SEQ ID NO:3, described in more detail below.

The adult female cDNA library was screened with an antisense probe,using stringent (i.e., standard) hybridization conditions as describedin Sambrook et al., ibid. The antisense probe, denoted GRF14, was basedon the DNA sequence derived from the amplified segment and has thesequence 5′ CTGTTTGAACCATAACATTATCAGATGG 3′ (SEQ ID NO:15). Plaqueswhich hybridized to the probe were rescreened, plaque purified andclones containing D. immitis nucleic acid sequence p22U (i.e., clonesthat hybridized with the antisense probe and having the apparent nucleicacid sequence designated in SEQ ID NO:3) were submitted to nucleic acidsequencing as described in Example 4.

An about 1016-nucleotide consensus sequence of D. immitis nucleic acidsequence p22U was determined and is presented as SEQ ID NO:3. Thededuced translation product is presented both with SEQ ID NO:3 and inSEQ ID NO:4. SEQ ID NO:3 apparently encodes a protein of about 208 aminoacids, the sequence including a stop codon spanning nucleotides about627 through about 629. There are two ATG codons spanning nucleotidesabout 39 to about 41 and spanning nucleotides about 57 to about 59.Although SEQ ID NO:3 encodes a protein of about the expected size (i.e.,predicted size of about 22 kD), the actual translation initiation siteof the protein is as yet unknown.

Nucleic acid sequences encoding all three partially sequenced trypticpeptides are included in SEQ ID NO:3, indicating that the sequence doesencode at least a portion of P22U. The portion of the 44 min trypticfragment that was sequenced spans amino acids about 77 to about 92 ofSEQ ID NO:4 and agrees with the derived sequence in all but one aminoacid. The portion of the 58 min tryptic fragment that was sequencedspans amino acids about 27 to about 48 of SEQ ID NO:4 and agrees withthe derived sequence in all but one amino acid. The portion of the 60min tryptic fragment that was sequenced spans amino acids about 145 toabout 166 of SEQ ID NO:4 and agrees with the derived sequence in all butone amino acid. A homology search of the non-redundant protein sequencedatabase was performed through the National Center for BiotechnologyInformation using the BLAST network. This database includesSwissProt+PIR+SPUpdate+GenPept+GPUpdate. The search was performed usingSEQ ID NO:4 and no significant homology with known proteins wasindicated.

Example 7

This example demonstrates the ability of D. immitis p22U to encode aprotein that selectively binds to immune serum.

Recombinant molecule pHis-p22U₆₀₈, containing D. immitis p22Unucleotides from about 41 through about 649 operatively linked to trctranscription control sequences and to a fusion sequence encoding apoly-histidine segment comprising 6 histidines was produced in thefollowing manner. An about 608-nucleotide DNA fragment containingnucleotides spanning from about 41 through about 649 of SEQ ID NO:3,called p22U₆₀₈, was PCR amplified from a clone containing D. immitisp22U using the primers 5′ GTTGCAAT-ATGGGATCCAATGAGCC 3′ (denoted SEQ IDNO:16 or 22USEN; BamHI site underlined) and 5′ CGCTAGTGCAGGATCCTCAATACTC3′ (denoted SEQ ID NO:17 or 22UANT; BamHI site underlined). The PCRproduct was digested with BamHI restriction endonuclease, gel purifiedand subcloned into expression vector pTrcHisB (available fromInvitrogen) that had been cleaved with BamHI. The resulting recombinantmolecule pHis-p22U₆₀₈ was transformed into E. coli to form recombinantcell E. coli:pHis-p22U₆₀₈. The recombinant cell was cultured in shakeflasks containing an enriched bacterial growth medium containing 0.1mg/ml ampicillin at about 37° C. When the cells reached an OD₆₀₀ ofabout 0.3, expression ofD. immitis p22U₆₀₈ was induced by addition ofabout 1 mM IPTG. Protein production was monitored by SDS PAGE ofrecombinant cell lysates, followed by Coomassie blue staining, usingstandard techniques. Recombinant cell E. coli:His-p22U₆₀₈ produced aprotein, denoted herein as PHIS-P22U₆₀₈, that migrated with an apparentmolecular weight of about 27 kD. Such a protein was not produced bycells transformed with the pTrcHisB plasmid lacking a D. immitis DNAinsert.

Immunoblot analysis of recombinant cell E. coli:pHis-p22U₆₀₈ lysatesindicates that the 22-kD protein is able to selectively bind to immunedog serum and, as such, is capable of binding to at least one componentof a serum that is capable of inhibiting D. immitis larval development.Immune dog serum essentially does not bind to lysates of cellstransformed with only the pTrcHisB plasmid.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. An isolated monoclonal antibody that selectively binds to a proteinconsisting of amino acid sequence SEQ ID NO:4.
 2. The antibody of claim1, wherein said protein selectively binds to immune serum that inhibitsD. immitis development.
 3. The antibody of claim 1, wherein saidantibody selectively binds to a protein encoded by a nucleic acidsequence SEQ ID NO:3.
 4. A composition comprising an excipient and anisolated monoclonal antibody that selectively binds to a proteinconsisting of amino acid sequence SEQ ID NO:4.
 5. The composition ofclaim 4, wherein said composition further comprises at least onecomponent selected from the group consisting of an adjuvant and acarrier.